A number of cancer-related autoimmune and neurologic diseases are associated with RNA-binding proteins. This revised application focuses on a structural (x-ray and NMR) and functional (impact of mutations) investigation of protein-RNA recognition in Fragile X mental retardation (FXMR) and paraneoplastic opsoclonus-myoclonus ataxia (POMA) syndromes. This project represents a collaborative effort with the Robert Darnell laboratory, which has biochemically identified the relevant protein-RNA complexes, and is currently addressing biological and clinical issues associated with these syndromes. Project 1: Our structural efforts on the FXMR syndrome have focused on the complex between an RGG peptide and a quadruplex-duplex neuronal RNA scaffold for which we have obtained exceptional NMR spectra, including spectra of samples uniformly 13C,15N-labeled in the peptide and RNA components. These structural efforts will be followed up by mutational experiments coupled with affinity measurements using surface plasmon resonance to identify energetic contributions involving key residues associated with molecular recognition. A new project involves the structure determination of a complex between one of the two FXMR KH domains and its in vitro-selected RNA target identified in the Darnell laboratory. We also propose to solve structures of three distinct sets of crystals of FXMR syndrome r(CGG)n repeats, n = 3, which have been grown in various space groups, one of which diffracts to 1.0 A resolution. Project 2: Our proposed structural studies of the POMA syndrome are directed towards our long-term goal of providing a structural understanding of how full length Nova (KH1-KH2-KH3) proteins target and regulate alternative splicing events within the alpha-2 glycine receptor subunit pre-mRNA, where the RNA target contains the UCAU-Y-UCAU-Y-UCAU sequence. We have already determined a crystal structure ol a Nova KH1/KH2 construct bound to its UCAN-UCAN-containing RNA hairpin, where KH1 targets one of the two UCAN RNA segments, while KH2 is involved in protein dimerization. We are currently mutating residues at the unanticipated KH dimeric interface to evaluate its functional role, as well as attempting to provide a molecular explanation for protein engineering efforts aimed at extending the KH-RNA interface.